Diagnosis of sleeping sickness depends on microscopic demonstration of trypanosomes in blood, cerebrospinal fluid (CSF) or lymph node aspirates. Parasitological methods are not easy to use in screening programmes. Development of serological tests for trypanosomiasis has been hindered by the ability of the parasite to keep changing its surface antigenic coat, which allows it to evade the host’s defense mechanisms. FIND is working with partners to develop serological methods based on either antibody or antigen detection that will be used as improved screening tools, or that would ideally be specific and sensitive enough to guide treatment.
Figure 1: The new HAT RDT developed by FIND and Standard Diagnostics. 20 µl of fresh blood from a finger prick is placed into the well of the RDT, followed by 4 drops of diluent. Results can be read after 15 minutes (view demo video). A positive sample has 2 or 3 red bands; a negative has one band (not shown in photo).
Antibody detection test
FIND and Standard Diagnostics (SD) have developed the first lateral flow rapid diagnostic test (RDT) for Trypanosoma brucei gambiense HAT that is cheap and easy to use for screening people (Figure 1). The tests are packed individually and are stable at a temperature of 40°C for up to 25 months; they are performed on fresh blood obtained from a finger prick, and no instrument or electricity is required. The RDT detects host antibodies to infection, and presence of parasites is confirmed by microscopy.
The new RDT is an alternative to the card agglutination test for trypanosomiasis (CATT), the primary screening tool used by control programmes in areas where T.b. gambiense HAT is endemic (Figure 2). Evaluation of prototypes of the new test at sites in Angola, Central African Republic and the Democratic Republic of the Congo (DRC) has been completed, with more than 14,000 participants being tested by both passive and active screening.
Figure 2: A technician takes blood by finger prick during a clinical evaluation of the new RDT test for sleeping sickness in Bandundu province, Democratic Republic of the Congo.
The results obtained have confirmed that the new RDT, which is the first ever to be developed for HAT, performs as well as CATT, with the additional advantages of simplicity, and not requiring refrigeration or any instruments. Based on these findings, the design of the product was locked and the test registered (Korean FDA and CE-marking).
Background on first generation antibody detection test
A study to evaluate the commercialized version of the rapid test was conducted in 2013 and 2014 at multiple sites in three endemic provinces of the Democratic Republic of the Congo. The objectives of this study were to confirm the accuracy of the test, determine the costs associated with its use, determine the cost-effectiveness of diagnostic strategies that use the rapid test and also determine its ease of use, in comparison to other methods. Results indicated that the sensitivity of the RDT was significantly higher than that of the screening test that is routinely used (CATT/T.b. gambiense), but that specificity was slightly lower. Publications including detailed results from this study are being finalized.
The new RDT has been developed with parasite antigens selected through a series of screening activities. Scientists and laboratories with antigens that were previously identified as candidate diagnostic probes have collaborated with FIND in screening native and recombinant proteins for their potential in diagnosis of both T.b. gambiense and T.b. rhodesiense sleeping sickness. In order to identify the most promising candidates, an initial panel of 35 different antigens was screened in a slot blot assay by MicroCoat Biotechnologie GmbH (Bernried, Germany), using a collection of well-characterized sera from patients infected with T.b. gambiense and T.b. rhodesiense, and from endemic controls. The first screen led to the selection of 18 antigens, which underwent two more rounds of screening using slot blot and ELISA, and new collections of clinical samples. Seven particularly promising antigens were transferred to Standard Diagnostics/Alere (South Korea), and two of them were finally selected for developing the new first generation RDT for T.b. gambiense HAT.
The RDT will be practical for integration into the horizontal health care system, at health facilities where single format tests such as malaria RDTs and HIV RDTs are already being used. Its inclusion in surveillance programs, in combination with other new methods such as LAMP, could provide an opportunity to develop cost-effective strategies for passive and active screening, thus increasing the coverage of the population at risk and accelerating the process of elimination of the disease.
Second generation antibody detection test
Meanwhile, FIND and partners have intensified efforts to develop a second generation RDT using recombinant antigens, which will be easier and cheaper to manufacture than the first generation RDT that is made of native antigens generated from pathogenic trypanosomes. A prototype has been developed, whose performance is being evaluated in a clinical trial that is being completed at multiple sites in the Democratic Republic of the Congo. As of January 2016, more than 250 HAT cases and 43,000 controls had been enrolled. While the final results of this study are expected to be available by the second quarter of 2016, an experts’ meeting was held at FIND on 14th January to review interim results (see photograph). The meeting concluded that the performance of the new test was comparable to the performance of the first generation RDT and therefore satisfactory, although the implementation strategies for this test remained to be defined. One option that was considered was to combine this new test with other screening tools to further improve sensitivity. The second generation RDT is planned to be made commercially available during the first half of 2016.
Antigen detection test
Identification of antibodies that are suitable for antigen detection assays and antigens for use in developing antibody detection tests have been running concurrently. FIND has been working with the Institute of Biotechnology at the University of Brussels, Belgium, to determine the feasibility of using camel heavy-chain antibodies (nanobodies) in tests to detect parasite antigens. A number of promising nanobodies have been identified and their potential for inclusion in an RDT has been explored. However, no convincing results have been obtained so far.
FIND has also worked with the Seattle Biomedical Research Institute (SBRI) to apply the single chain variable fragment (scFv) antibody engineering technology in development of optimized antibody probes for trypanosome antigens in blood. Using a technology called yeast display, high-affinity antibody fragments for a number of T. brucei proteins were generated, and those that were best suited for diagnostic detection in human samples identified. The sensitivity and stability of the probes for the chosen antigens was enhanced further by antibody engineering methods. Although a set of antibody probes with promising characteristics of sensitivity, stability, and manufacturability was obtained, none of these probes were sufficiently convincing to be taken further into test development.